1.1 Field of the Invention
The present invention relates generally to the field of oncology. More particularly, certain embodiments concern methods for making and using lipid-carrier protein conjugate compositions such as phosphatidylserine (PS)-conjugates for generating lipid-specific immune responses in an animal. Also disclosed are methods for making PS antigen and antibody compositions and their use in a variety of therapeutic applications, including the formulation of pharmaceutical compositions for the prevention and treatment of cancers.
1.2 Description of the Related Art
The results of many studies have led to the concept that membrane phospholipid asymmetry is ubiquitous. The outer leaflet of eukaryotic plasma membranes contains most of the cholinephospholipids, whereas the aminophospholipids are mainly present in the cell""s inner leaflet (Devaux, 1991; Schroit and Zwaal, 1991). While asymmetry seems to be the rule for normal cells, loss of membrane lipid sidedness, in particular the emergence of phosphatidylserine (PS) at the cell surface, results in the expression of altered surface properties that modulates cell function and influences the cells interaction with its environment (Zwaal and Schroit, 1997). For example, the exposure of PS promotes coagulation and thrombosis by platelets (Bevers et al., 1983; Rosing et al., 1985; Thiagarajan and Tait, 1990) and the recognition of apoptotic (Fadok et al., 1992; Bennett et al., 1995; Sambrano and Steinberg, 1995; Verhoven et al., 1995) and aged (Herrmann and Devaux, 1990; Geldwerth et al., 1993; Connor et al. 1994) cells by the reticuloendothelial system.
To characterize these and other PS-related processes, new tools are required to determine physiologically-dependent alterations in the distribution of PS in cell membranes. Although the application of classical biochemical methodologies (Gordesky et al., 1975; Schick et al., 1976; Etemadi, 1980; Bevers et al., 1982) has yielded important information on PS asymmetry, most of these methods are invasive and destructive. Recently developed methods, such as the PS-dependent prothrombinase assay (Bevers et al., 1983; Rosing et al., 1980; Van Dieijen et al., 1981) and labeled annexin V binding (Thiagarajan and Tait, 1990; Tait and Gibson, 199,4; Vermes et al., 1995; Kuypers et al., 1996), are non-invasive and have provided the means to assess the presence and topology of PS in the outer leaflet of viable cells. These methods, however, require the inclusion of various plasma cofactors and/or divalent cations which might influence the lateral distribution of lipids in the plane of the membrane.
While antibodies against different membrane components has become an indispensable aid in the study of membrane structure and function, little attention has been given to the application of lipid-specific antibodies for studying lipid-dependent processes. Because of the inherent difficulty of producing antibodies against small highly conserved lipids, the development of lipid antibodies has progressed slowly. Nonetheless, several laboratories have produced antibodies against certain phospholipid species by immunization with liposomes (Maneta-Peyret et al., 1988; Maneta-Peyret et al., 1989; Banedi and Alving, 1990) or by adsorption of monomeric phospholipids to proteins (Maneta-Peyret et al., 1989; Tamamura et al., 1971), bacteria (Umeda et al., 1989) and acrylamide (Maneta-Peyret et al., 1988; Maneta-Peyret et al., 1989). Antibodies produced by these methods, however, may cross-react with different lipids (Banedi and Alving, 1990; Umeda et al., 1989) and other phosphate-containing moieties (Alving, 1986).
1.3 Deficiencies in the Prior Art
While some methods have been developed in these areas, what is lacking in the prior art are effective methodologies for generating immune responses that are useful in various treatment regimens, including those specific for oncology.
Several reports on the production of PS antibodies have been published. These include unrelated methods and approaches using PS-containing liposomes (Banedji and Alving, 1990), PS-coated Salmonella (Umeda et at., 1989) and acrylamide-entrapped PS (Maneta-Peyret et al., 1988). There is one report concerning carrier (KLH)-coupled PS (Bate et al., 1993). However, the chemistry employed to make the conjugate couples lipid to the protein carrier via the lipids primary amine thereby destroying antigenic specificity. Immunization with this conjugate produced antibody activity which inhibited the production of tumor necrosis factor by malaria-infected erythrocytes. Whether the antibodies had any lipid specificity was not investigated. This is unlikely, however, considering that the conjugation chemistry destroyed the key primary amine determinant of phosphatidylserine. Thus, there exists an immediate need for an effective method of producing highly-specific anti-PS antibodies and cell-mediated PS responses for use in the diagnosis and treatment of various cancers and related conditions.
The present invention overcomes one or more of these and other drawbacks inherent in the prior art by providing novel compositions and methods for their use in the induction of an autoimmune response to lipids such as PS. Disclosed are methods for the preparation and use of novel lipid antigen compositions which generate an immune response in an animal. Also disclosed are methods for the use of lipid-specific antibody compositions, including those specific for PS, in a variety of diagnostic and therapeutic regimens, including the treatment of cancer.
Exemplary preferred methods and compositions according to this invention, which will be described in greater detail in the remainder of the invention include:
Methods for inhibiting cancer cell growth or killing cancer cells, comprising eliciting an immune response with an immunologically effective amount of a composition comprising a phosphatidylserine/polypeptide conjugate;
Methods for treating cancer comprising eliciting an immune response with an immunologically effective amount of a composition comprising a phosphatidylserine/polypeptide conjugate;
Methods for treating cancer comprising contacting a subject with a lipid or lipid/polypeptide conjugate effective to treat said cancer;
Methods of generating an immune response, comprising administering to an animal a pharmaceutical composition comprising an immunologically effective amount of a phosphatidylcholine/polypeptide or a phosphatidylserine/polypeptide conjugate composition;
Methods for treating cancer in an animal, comprising generating in said animal an immune response to a composition comprising a phosphatidyiserine or phosphatidylserine/polypeptide conjugate effective to treat said cancer,
Methods of making an antibody that specifically binds to phosphatidylserine or a phosphatidylcholine/polypeptide or a phosphatidylserine/polypeptide conjugate, said methods comprising administering to an animal a pharmaceutical composition comprising an immunologically effective amount of a phosphatidylcholine/polypeptide or a phosphatidylserine/polypeptide conjugate composition. Presently preferred conjugates for use in such methods are, for example, phosphatidylserine/BSA, phosphatidyiserine/KLR phosphatidylserine/BGG, and phosphatidylserine/xcex22-glycoprotein I conjugate;
Antibodies that specifically bind to phosphatidylserine or a phosphatidylcholine/polypeptide or a phosphatidylserine/polypeptide conjugate, said antibody made by a process comprising administering to an animal a pharmaceutical composition comprising an immunologically effective amount of a phosphatidylcholine/polypeptide or a phosphatidyiserine/polypeptide conjugate composition. Presently preferred conjugates for use in such processes are, for example, phosphatidylserine/BSA, phosphatidylserine/KLH, phosphatidylserine/BGG, and phosphatidylserine/xcex22-glycoprotein I conjugate;
Methods for detecting a phosphatidylserine, phosphatidylcholine/polypeptide or a phosphatidylserine/polypeptide conjugate in a biological sample, comprising the steps of:
(a) obtaining a biological sample suspected of containing a phosphatidylcholine/polypeptide or a phosphatidylserine/polypeptide conjugate;
(b) contacting said sample with a first antibody that binds to a phosphatidylcholine/polypeptide or a phosphatidylserine/polypeptide conjugate, under conditions effective to allow the formation of immune complexes; and
(c) detecting the immune complexes so formed; and
Immunodetection kits comprising, in suitable container means, an antibody that specifically binds to phosphatidylserine or to a phosphatidylserine/polypeptide conjugate, and an immunodetection reagent.
In an important embodiment, the invention provides antigenic PS conjugate compositions and means for making and using these compositions. In the context of this invention, a PS composition is understood to comprise one or more phosphatidylserine compositions that are able to generate an immune response in an animal. A PS antibody composition is understood to mean an antibody which is specific for PS. Preferably, the antigen composition comprises a lipid-carrier protein conjugate. The carrier protein may be maleimide-activated, or alternatively, may be prepared by introduction of reactive sulfhydryls into the carrier protein. Alternatively, one may prepare proteins by non-covalent electrostatic interactions between negatively-charged anionic phospholipids and lipid binding proteins such as xcex22-glycoprotein I, also known as apolipoprotein H. Exemplary carrier proteins contemplated to be useful in the present methods include various commonly used carrier proteins including BSA (bovine serum albumin), KLH (keyhole limpet hemocyanin), BGG (bovine gamma globulin) and diphtheria toxin. As such, a PS composition of the present invention is also understood to comprise one or more PS-containing or other negatively charged formulations that elicit an immune response in an animal.
In a preferred embodiment, administration of a therapeutically effective dose of a lipid-conjugate antigen composition, such as a PS-conjugate to an animal induces in the animal antibodies which are specific for the particular lipid. In one embodiment, the carrier protein is a glycoprotein, such as xcex22-glycoprotein I.
In certain aspects, the present invention concerns novel lipid-carrier antigen compositions which evoke a specific immune response to the lipid. In particular, PS antigen compositions have been developed which have shown remarkable utility both in vitro and in vivo. In particular, PS antigen compositions have been produced to provide vaccine or therapeutic compositions useful in the prevention or treatment of various cancers, such as lymphomas and renal and bladder cancers.
A further aspect of the invention is the preparation of immunological compositions comprising both antibody and cell-mediated immune responses for diagnostic and therapeutic methods relating to the detection and treatment of a variety of cancers and related illnesses.
The invention also encompasses PS antigen and antibody compositions together with pharmaceutically-acceptable excipients, carriers, diluents, adjuvants, and other components, such as peptides, antigens, or pharmaceuticals, as may be employed in the formulation of particular vaccines or antibody compositions.
Antibodies may be of several types including those raised in heterologous donor animals or human volunteers immunized with PS compositions, monoclonal antibodies (mAbs) resulting from hybridomas derived from fuisions of B cells from PS-immunized animals or humans with compatible myeloma cell lines, so-called xe2x80x9chumanizedxe2x80x9d mAbs resulting from expression of gene fusions of combinatorial determining regions of mAb-encoding genes from heterologous species with genes encoding human antibodies, or PS-reactive antibody-containing fractions of plasma from human or animal donors.
Also disclosed is a method of generating an immune response in an animal. The method generally involves administering to an animal a pharmaceutical composition comprising an immunologically effective amount of a PS composition disclosed herein Preferred animals include mammals, and particularly humans. Other preferred animals include murines, bovines, equines, ovines, caprines, opines, porcines, canines, felines, and the like. The composition may include partially or significantly purified PS antigen compositions, and particularly will include one or more of the PS conjugate compositions described herein.
By xe2x80x9cimmunologically effective amountxe2x80x9d is meant an amount of a peptide/lipid composition that is capable of generating an immune response in the recipient animal. This includes both the generation of an antibody response (B cell response), and/or the stimulation of a cytotoxic immune response (T cell response). The generation of such an immune response will have utility in both the production of useful bioreagents, e.g., CTLs and, more particularly, reactive antibodies, for use in diagnostic embodiments, and will also have utility in various prophylactic and therapeutic embodiments.
Immunoformulations of this invention, whether intended for vaccination, treatment, or for the generation of antibodies useful in the detection of PS or other lipids may comprise native, or synthetically-derived PS antigenic compositions produced using the methods described herein. As such, antigenic functional equivalents of the PS compositions described herein also fall within the scope of the present invention. An xe2x80x9cantigenically functional equivalentxe2x80x9d protein or peptide is one that incorporates an epitope that is immunologically cross-reactive with one or more epitopes derived from any of the particular PS compositions disclosed herein. Antigenically functional equivalents, or epitopic sequences and lipid formulations, may be first designed or predicted and then tested, or may simply be directly tested for cross-reactivity. Also encompassed by the invention are modified PS-conjugates which have improved antigenicity or other desirable characteristics, and that are produced in a fashion similar to those described herein.
In still further embodiments, the present invention concerns immunodetection methods and associated kits. It is contemplated that the PS antigen compositions, and particularly PS conjugates, may be employed to detect antibodies having reactivity therewith, or, alternatively, antibodies prepared in accordance with the present invention, may be employed to detect PS-containing cells, compositions, tissues, and the like. Either type of kit may be used in the immunodetection of compounds, present within clinical samples. The kits may also be used in antigen or antibody purification, as appropriate.
In general, the preferred immunodetection methods will include first obtaining a sample suspected of containing a lipid-specific antibody, such as a biological sample from a patient, and contacting the sample with a first lipid and/or lipid conjugate antigen composition under conditions effective to allow the formation of an immunocomplex (primary immune complex). One then detects the presence of any primary immnunocomplexes that are formed.
Contacting the chosen sample with the lipid antigen composition under conditions effective to allow the formation of (primary) immune complexes is generally a matter of simply adding the antigen composition to the sample. One then incubates the mixture for a period of time sufficient to allow the added antigens to form immune complexes with, i.e., to bind to, any antibodies present within the sample. After this time, the sample composition, such as a tissue section, ELISA plate, dot blot or western blot, will generally be washed to remove any non-specifically bound antigen species, allowing only those specifically bound species within the immune complexes to be detected.
The detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches known to the skilled artisan and described in various publications, such as, e.g., Nakamura et al. (1987), incorporated herein by reference. Detection of primary immune complexes is generally based upon the detection of a label or marker, such as a radioactive, fluorescent, biological or enzymatic label, with enzyme tags such as alkaline phosphatase, urease, horseradish peroxidase and glucose oxidase being suitable. The particular antigen employed may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of bound antigen present in the composition to be determined.
Alternatively, the primary immune complexes may be detected by means of a second binding ligand that is linked to a detectable label and that has binding affinity for the first protein or peptide. The second binding ligand is itself often an antibody, which may thus be termed a xe2x80x9csecondaryxe2x80x9d antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies and the remaining bound label is then detected.
For diagnostic purposes, it is proposed that virtually any sample suspected of containing the antibodies of interest may be employed. Exemplary samples include clinical samples obtained from a patient such as blood or serum samples, cerebrospinal, synovial, or bronchoalveolar fluid, ear swabs, sputum samples, middle ear fluid or even perhaps urine samples may be employed. Such methods may be useful for the diagnosis and treatment of various cellular disorders, and in particular, cancers and related conditions.
Furthermore, it is contemplated that such embodiments may have application to nonclinical samples, such as in the titering of antibody samples, in the selection of hybridomas, and the like. Alternatively, the clinical samples may be from veterinary sources and may include such domestic animals as cattle, sheep, and goats. Samples from murine, ovine, opine, caprine, feline, canine, and equine sources may also be used in accordance with the methods described herein.
In related embodiments, the present invention contemplates the preparation of kits that may be employed to detect the presence of PS-specific antibodies in a sample. Generally speaking, kits in accordance with the present invention will include a suitable lipid, lipid/protein or peptide together with an immunodetection reagent, and a means for containing the lipid, protein or peptide and reagent.
The immunodetection reagent will typically comprise a label associated with a PS antigen composition, or associated with a secondary binding ligand. Exemplary ligands might include a secondary antibody or lipid binding protein directed against the first PS antigen or antibody composition, or a biotin or avidin (or streptavidin) ligand having an associated label. Detectable labels linked to antibodies that have binding affinity for a human antibody are also contemplated, e.g., for protocols where the first reagent is a PS antigen composition that is used to bind to a reactive antibody from a human sample. Of course, as noted above, a number of exemplary labels are known in the art and all such labels may be employed in connection with the present invention. The kits may contain antigen, lipid binding protein, or antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit.
The container means will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antigen may be placed, and preferably suitably allocated. Where a second binding ligand is provided, the kit will also generally contain a second vial or other container into which this ligand or antibody may be placed. The kits of the present invention will also typically include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained.
Another aspect of the invention are immunodetection kits containing lipid or lipid-carrier conjugate antigen-specific antibodies and suitable immunodetection reagents such as a detectable label linked to a protein, peptide or the antibody itself. Alternatively, the detectable label may be linked to a second antibody which binds to a lipid-specific antibody as disclosed herein.
Related embodiments include diagnostic and therapeutic kits which include pharmaceutically-acceptable formulations of either the antibodies, lipid, lipid/peptide, or peptide antigens disclosed herein. Such kits are useful in the detection of lipids such as PS in clinical samples, and also useful for promoting an immune response in an animal, and in the formulation of vaccine compositions effective in the treatment of a variety of cancers.
In certain embodiments, the inventor contemplates the use of the lipid-carrier conjugate compositions for the preparation of anti-cancer vaccines or treatment regimens for administration to an animal, and in particular, a human. It is expected that to achieve an xe2x80x9cimmunologically effective formulationxe2x80x9d it may be desirable to administer a lipid-carrier conjugate composition, such as a PS-carrier antigen composition, to the human or animal subject in a pharmaceutically acceptable composition comprising an immunologically effective amount of an antigen composition mixed with other excipients, carriers, or diluents which may improve or otherwise alter stimulation of B cell and/or T cell responses, or immunologically inert salts, organic acids and bases, carbohydrates, and the like, which promote stability of such mixtures. Immunostimulatory excipients, often referred to as adjuvants, may include salts of aluminum (often referred to as Alums), simple or complex fatty acids and sterol compounds, physiologically acceptable oils, polymeric carbohydrates, chemically or genetically modified protein toxins, and various particulate or emulsified combinations thereof. Lipid conjugate antigen compositions within these mixtures, or each variant if more than one are present, would be expected to comprise about 0.0001 to 1.0 milligrams, or more preferably about 0.001 to 0.1 milligrams, or even more preferably less than 0.1 milligrams per dose.
A therapeutic kit comprising, in suitable container means, one or more lipid-conjugate antigen(s) or antibody composition(s) of the present invention in a pharmaceutically acceptable formulation, represents another important aspect of the invention.
The kit may comprise a single container means that contains the lipid-conjugate antigen(s) or antibody composition(s). The container means may, if desired, contain a pharmaceutically acceptable sterile excipient, having associated with it, the lipid-conjugate antigen(s) or antibody composition(s) and, optionally, a detectable label or imaging agent. The formulation may be in the form of a gelatinous composition (e.g., a collagenous composition), a powder, solution, matrix, lyophilized reagent, or any other such suitable means. In certain cases, the container means may itself be a syringe, pipette, or other such like apparatus, from which the lipid-conjugate antigen(s) or antibody composition(s) may be applied to a tissue site, tumor, skin lesion, wound area, or other site of administration. However, the single container means may contain a dry, or lyophilized, mixture of one or more lipid-conjugate antigen(s) or antibody composition(s), which may or may not require pre-wetting before use.
Alternatively, the kits of the invention may comprise distinct container means for each component. In such cases, one or more containers would contain each of the PS composition(s), either as sterile solutions, powders, lyophilized forms, etc., and the other container(s) would include a matrix, solution, or other suitable delivery device for applying the composition to the body, bloodstream, or to a tissue site, skin lesion, tumor cell, wound area, or other site of administration. Such delivery device may or may not itself contain a sterile solution, diluent, gelatinous matrix, carrier or other pharmaceutically-acceptable components.
The kits may also comprise a second or third container means for containing a sterile, pharmaceutically acceptable buffer, diluent or solvent. Such a solution may be required to formulate the lipid-conjugate antigen(s) or antibody composition(s) into a more suitable form for application to the body, e.g., as a topical preparation, or alternatively, in oral, parenteral, or intravenous forms. It should be noted, however, that all components of a kit could be supplied in a dry form (lyophilized), which would allow for xe2x80x9cwettingxe2x80x9d upon contact with body fluids. Thus, the presence of any type of pharmaceutically acceptable buffer or solvent is not a requirement for the kits of the invention. The kits may also comprise a second or third container means for containing a pharmaceutically acceptable detectable imaging agent or composition.
The container means will generally be a container such as a vial, test tube, flask, bottle, syringe or other container means, into which the components of the kit may placed. The components may also be aliquoted into smaller containers, should this be desired. The kits of the present invention may also include a means for containing the individual containers in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vials or syringes are retained.
Irrespective of the number of containers, the kits of the invention may also comprise, or be packaged with, an instrument for assisting with the placement of the lipid-carrier conjugate, or antibodies reactive therewith, within the body of an animal. Such an instrument may be a syringe, needle, surgical instrument, pipette, forceps, or any such medically approved delivery vehicle.
As described above, an important embodiment of the invention is the formulation of lipid-specific antibodies which are useful in detecting and treating various cancers in an animal, and particularly, in a human. Means for preparing and characterizing antibodies are well known in the art (See, e.g., Harlow and Lane (1988); incorporated herein by reference). The methods for generating mAbs generally begin along the same lines as those for preparing polyclonal antibodies. Briefly, a polyclonal antibody is prepared by immunizing an animal with one or more of the lipid-carrier protein compositions disclosed herein and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
As is well known in the art, a given composition may vary in its immunogenicity. With respect to preparing lipid-specific antibodies, it is necessary to boost the host immune system, and may be achieved by coupling the lipid of interest, such as PS, to a carrier. As described above, exemplary and preferred carriers include polypeptide carriers such as KLH, BSA, and xcex22-glycoprotein I. Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers, as well as bovine gamma globulin and/or diphtheria toxoid. Although means for conjugating lipids to a carrier protein are well-known in the art, two particular synthesis methods are disclosed herein which have been particularly useful in preparing covalent lipid-specific antibody formulations.
mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Pat. No. 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep or frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.
Following immunization, somatic cells with the potential for producing antibodies, specifically B-lymphocytes (B-cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately about 5xc3x97107 to about 2xc3x97108 lymphocytes.
The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fuision efficiency, and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1986; Campbell, 1984). For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.
One preferred murine myeloma cell is the NS-1 myeloma cell line (also termed P3-NS-1-Ag4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.
Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described (Kohler and Mistein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (vol./vol.) PEG, by Gefter et al. (1977). The use of electrically induced fusion methods is also appropriate (Goding, 1986).
Fusion procedures usually produce viable hybrids at low frequencies, about 1xc3x9710xe2x88x926 to about 1xc3x9710xe2x88x928. However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.
The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.
This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimnmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for mAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fuision. The injected animal develops tumors secreting the specific mAb produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide mabs in high concentration. The individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.